VWF is a multimeric adhesive glycoprotein present in the plasma of mammals, which has multiple physiological functions. During primary hemostasis VWF acts as a mediator between specific receptors on the platelet surface and components of the extracellular matrix such as collagen. Via the GP IIbIIIa receptor, VWF also contributes to hemostasis also via promoting platelet-platelet interaction. Moreover, VWF serves as a carrier and stabilizing protein for procoagulant FVIII. VWF is synthesized in endothelial cells and megakaryocytes as a 2813 amino acid precursor molecule. The precursor polypeptide, pre-pro-VWF, consists of a 22-residue signal peptide, a 741-residue pro-peptide and the 2050-residue polypeptide found in mature plasma VWF (Fischer et al., FEBS Lett. 351: 345-348, 1994). Upon secretion into plasma VWF circulates in the form of various species with different molecular sizes. These VWF molecules consist of oligo- and multimers of the mature subunit of 2050 amino acid residues. VWF can be usually found in plasma as one dimer up to multimers consisting of 50-100 dimers (Ruggeri et al. Thromb. Haemost. 82: 576-584, 1999). The in vivo half-life of human VWF in the human circulation is approximately 12 to 20 hours.
The most frequent inherited bleeding disorder in humans is von Willebrand's disease (VWD), which can be treated by replacement therapy with concentrates containing VWF of plasmatic or recombinant origin.
VWF can be prepared from human plasma as for example described in EP 05503991. EP 0784632 describes a method for isolating recombinant VWF.
VWF is known to stabilize FVIII in vivo and, thus, plays a crucial role to regulate plasma levels of FVIII and as a consequence is a central factor to control primary and secondary hemostasis. It is also known that after intravenous administration pharmaceutical preparations containing VWF in VWD patients an increase in endogenous FVIII:C to 1 to 3 units per ml in 24 hours can be observed demonstrating the in vivo stabilizing effect of VWF on FVIII.
FVIII is a blood plasma glycoprotein of about 260 kDa molecular mass, produced in the liver of mammals. It is a critical component of the cascade of coagulation reactions that lead to blood clotting. Within this cascade is a step in which factor IXa (FIXa), in conjunction with FVIII, converts factor X (FX) to an activated form, FXa. FVIII acts as a cofactor at this step, being required with calcium ions and phospholipid for the activity of FIXa. The most common hemophilic disorders is caused by a deficiency of functional FVIII called hemophilia A.
An important advance in the treatment of hemophilia A has been the isolation of cDNA clones encoding the complete 2,351 amino acid sequence of human FVIII (U.S. Pat. No. 4,757,006) and the provision of the human FVIII gene DNA sequence and recombinant methods for its production.
Analysis of the deduced primary amino acid sequence of human FVIII determined from the cloned cDNA indicates that it is a heterodimer processed from a larger precursor polypeptide. The heterodimer consists of a C-terminal light chain of about 80 kDa in a metal ion-dependent association with an about 210 kDa N-terminal heavy chain fragment. (See review by Kaufman, Transfusion Med. Revs. 6:235 (1992)). Physiological activation of the heterodimer occurs through proteolytic cleavage of the protein chains by thrombin. Thrombin cleaves the heavy chain to a 90 kDa protein, and then to 54 kDa and 44 kDa fragments. Thrombin also cleaves the 80 kDa light chain to a 72 kDa protein. It is the latter protein, and the two heavy chain fragments (54 kDa and 44 kDa above), held together by calcium ions, that constitute active FVIII. Inactivation occurs when the 72 kDa and 54 kDa proteins are further cleaved by thrombin, activated protein C or FXa. In plasma, this FVIII complex is stabilized by association with a 50-fold excess of VWF protein (“VWF”), which appears to inhibit proteolytic destruction of FVIII as described above.
The amino acid sequence of FVIII is organized into three structural domains: a triplicated A domain of 330 amino acids, a single B domain of 980 amino acids, and a duplicated C domain of 150 amino acids. The B domain has no homology to other proteins and provides 18 of the 25 potential asparagine(N)-linked glycosylation sites of this protein. The B domain has apparently no function in coagulation and can be deleted with the B-domain deleted FVIII molecule still having procoagulatory activity.
The stabilizing effect of VWF on FVIII has also been used to aid recombinant expression of FVIII in CHO cells (Kaufman et al. 1989, Mol Cell Biol).
Until today the standard treatment of Hemophilia A and VWD involves frequent intravenous infusions of preparations of FVIII and VWF concentrates derived from the plasmas of human donors or in case of FVIII that of pharmaceutical preparations based on recombinant FVIII. While these replacement therapies are generally effective, e.g. in severe hemophilia A patients undergoing prophylactic treatment, Factor VIII has to be administered intravenously (i.v.) about 3 times per week due to the short plasma half life of Factor VIII of about 12 hours. Already if levels of above 1% of the FVIII activity in healthy non-hemophiliacs is reached, e.g. by a raise of FVIII levels by 0.01 U/ml, severe hemophilia A is turned into moderate hemophilia A. In prophylactic therapy dosing regimes are designed such that the trough levels of FVIII activity do not fall below levels of 2-3% of the FVIII activity in healthy non-hemophiliacs. Each i.v. administration is cumbersome, associated with pain and entails the risk of an infection especially as this is mostly done in home treatment by the patients themselves or by the parents of children being diagnosed for hemophilia A. In addition the frequent i.v. injections inevitably result in scar formation, interfering with future infusions. As prophylactic treatment in severe hemophilia is started early in life, with children often being less than 2 years old, it is even more difficult to inject FVIII 3 times per week into the veins of such small patients. For a limited period, implantation of port systems may offer an alternative. Despite the fact that repeated infections may occur and ports can cause inconvenience during physical exercise, they are nevertheless typically considered to be favorable as compared to intravenous injections.
Thus there is a great medical need to obviate the need to infuse VWF or FVIII intravenously.
As FVIII is a very large and labile molecule it exhibits a very low bioavailability due to insufficient absorption and severe degradation, if given subcutaneously, intramuscularly or intradermally, i.e. extravascularly.
EP0710114 discloses that FVIII formulations of a B-domain deleted FVIII in a concentration above 1000 IU/ml are suitable for subcutaneous, intramuscular or intradermal administration, leading to a bioavailability of 5-10% after s.c. administration in monkeys measuring the area under the activity (FVIII:C)-time curve.
EP0772452 discloses that FVIII formulations of a B-domain deleted FVIII in a concentration of at least 500 IU/ml together with an organic additive when administered subcutaneously can lead for more than 6 hours to a FVIII plasma level of at least 1.5% of normal FVIII levels. Using hydrolyzed gelatin or soybean oil emulsion as the organic additive and a B-domain deleted FVIII in a concentration of 1100 IU/ml and a dose of 10000 IU/kg, more than 50% bioavailability as measured as the area under the activity (FVIII:C)-time curve was seen in cynomolgus monkeys.
EP1258497 discloses a bioavailability of 5.3% when a B-domain deleted FVIII (specific activity 15000 IU/mg; dose 2500 IU/kg) was administered subcutaneously, whereas a PEGylated conjugate of FVIII achieved bioavailabilities of 22% and 19% respectively in cynomolgus monkeys.
EP 0871649 and EP1258497 disclose an increase of the bioavailability of a pegylated B-domain deleted FVIII and additionally propose to stabilize VWF and/or a combination of FVIII and VWF by conjugation to enable subcutaneous, intramuscular or intradermal administration to treat hemophilia A or VWD respectively.
WO 2006/071801 teaches the pegylation of VWF which may be administered by injection, such as intravenous, intramuscular, or intraperitoneal injection.
It has now been surprisingly found that, although being an extraordinarily large molecule (VWF multimers range from 1 MDa to 20 MDa) VWF can be taken up into the blood stream when administered extravascularly even without any stabilizing covalent modifications, which can entail an increased risk of immune responses, and that VWF can be used to enhance the uptake of FVIII when co-administered with FVIII non-intravenously.
The present invention thus relates to a composition suitable for extravascular administration in the therapy of von Willebrand disease (VWD) and/or hemophilia A comprising von Willebrand factor (VWF)
The ratio of VWF over FVIII is to be understood in the sense of the invention to be the ratio of VWF antigen units over FVIII activity units.
The VWF antigen (VWF:Ag) can be quantified by various immunologic assays, with the most frequently used are Laurell rocket electrophoresis, electroimmunoassay and enzyme-linked immunosorbent assay (ELISA) [Dalton & Savidge, 1989]. Dalton R G, Savidge G F. Progress in vWf methodology and its relevance in VWD. In: Seghatchian M J & Savidge G F (eds): Factor VIII—von Willebrand Factor, CRC Press, Inc., Boca Raton, Fla. 1989, Vol. I, pp. 129-145. Applying the same standard as reference all these, commercially available, tests, generate essentially identical results.
Factor VIII activity can be determined by a one-stage assay (measuring fibrin formation time in one single reaction step (Rizza et al. 1982. Coagulation assay of FVIII:C and FIXa in Bloom ed. The Hemophilias. NY Churchill Livingston 1992)) or a chromogenic assay (the speed with which an enzyme forms is measured by using the enzyme for the splitting of a chromogenic substrate (S. Rosen, 1984. Scand J Haematol 33: 139-145, suppl.)). Both approaches generate essentially identical findings and are also identically named FVIII:C
For both VWF:Ag and FVIII:C one international unit (IU) is defined by the current international standard established by the World Health Organization, with one IU FVIII:C or VWF:Ag is approximately equal to the level of Factor VIII or VWF found in 1.0 mL of fresh-pooled human plasma.
Another embodiment of the invention is the use of a pharmaceutical composition suitable for extravascular administration in the therapy of von Willebrand disease (VWD) and/or hemophilia A comprising von Willebrand factor (VWF) or a pharmaceutical composition comprising FVIII and VWF wherein the ratio of VWF antigen over FVIII activity is larger than 2:1. For the manufacture of a medicament for the treatment of VWD and/or hemophilia A when administered extravascularly.
By way of non-limiting example the ratio of VWF antigen to FVIII activity can be more than 2:1 preferentially more than 3:1, more preferentially more than 5:1, even more preferentially more than 15:1 and most preferentially more than 25:1.
Also encompassed by the invention is the use of VWF for the manufacture of a medicament to treat VWD and/or hemophilia A wherein after extravascular co-administration with a pharmaceutical preparation of FVIII either                a) the time period during which the FVIII activity in plasma is increased by at least 0.01 U/ml after injection is prolonged, preferably by a factor of 3, more preferably by a factor of 5, most preferably by a factor of 10        or        b) the maximal concentration of FVIII activity in plasma is increased, preferably by 3 fold, more preferably by 10 fold, most preferably by 20 fold        or        c) the area under the data curve (AUDC) of FVIII activity is increased, preferably by 5 fold, more preferably by 15, most preferably by 30 fold.as compared to the respective parameter when said pharmaceutical composition of FVIII is administered in the same concentration, dose and in the same mode of extravascular administration but without VWF.        
Preferentially purified VWF is used. Purified VWF in the sense of the invention encompasses VWF compositions in which VWF:Ag is present in a liquid or if stored lyophilized in the liquid after reconstitution prior to injection at a concentration which is by at least a factor of 20, preferentially by at least a factor of 75, more preferentially by at least a factor of 150 higher as compared to its concentration in plasma. Preferably resuspended cryoprecipitate and other low purity preparations of VWF are not used and the purified VWF is enriched to higher purity than in cryoprecipitate. Preferentially VWF of a purity of more than 1 U VWF:Ag/mg total protein (without added stabilizing proteins), more preferentially VWF of a purity of more than 10 U VWF:Ag/mg total protein (without added stabilizing proteins), even more preferentially VWF of a purity of more than 25 U VWF:Ag/mg total protein (without added stabilizing proteins) is used.
Preferentially purified FVIII is used. Purified FVIII in the sense of the invention encompasses FVIII compositions in which FVIII:C is present in a liquid or if stored lyophilized in the liquid after reconstitution prior to injection by at least a factor of 10, preferentially by at least a factor of 30, more preferentially by at least a factor of 70 as compared to its concentration in plasma. Preferably resuspended cryoprecipitate is not used and the purified FVIII is enriched to higher to higher purity than FVIII in cryoprecipitate. Preferentially FVIII of a specific activity (FVIII:C/mg total protein without added stabilizing proteins) of 1 IU/mg or more preferentially more than 5 IU/mg or even more preferentially more than 10 IU/mg is used.
Preferably the formulation comprising VWF or VWF in combination with FVIII is administered subcutaneously. However all other modes of extravascular administration are encompassed, e.g. intramuscular or intradermal administration.
By way of non-limiting example the concentration of VWF can be equal to or more than 150 U (VWF:Ag)/mL preferentially equal to or more 450 U (VWF:Ag)/mL, most preferentially equal to or more than 1500 U (VWF:Ag)/mL.
A typical dose could be equal to or more than 225 U (VWF:Ag)/kg or equal to or more than 75 U (VWF:Ag)/kg, or equal to or more 15 U (VWF:Ag)/kg.
By way of non-limiting example a typical dose of FVIII activity could be equal to or more than 75 U/kg or equal to or more than 25 U/kg or equal to or more than 5 U/kg.
The source of VWF or FVIII is irrelevant, e.g. it can be derived from human plasma or can be produced recombinantly.
When FVIII is recombinant, it can be either in its full-length form or preferably a deletion derivative thereof. More preferably the deletion derivative is recombinant factor VIII SQ (r-VIII SQ). By deletion derivative is here meant coagulation factor VIII, in which the whole or part of the B-domain is missing. Additionally, the factor VIII molecule, and in particular the r-VIII SQ molecule, can be chemically modified, e.g. by PEGylation, covalently linked carbohydrates or polypeptides, in order to improve the stability of the molecule in vivo.
The invention further relates to polynucleotides encoding a modified VWF or FVIII as described in this application. The term “polynucleotide(s)” generally refers to any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA or modified RNA or DNA. The polynucleotide may be single- or double-stranded DNA, single or double-stranded RNA. As used herein, the term “polynucleotide(s)” also includes DNAs or RNAs that comprise one or more modified bases and/or unusual bases, such as inosine. It will be appreciated that a variety of modifications may be made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term “polynucleotide(s)” as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells.
The skilled person will understand that, due to the degeneracy of the genetic code, a given polypeptide can be encoded by different polynucleotides. These “variants” are encompassed by this invention.
Preferably, the polynucleotide of the invention is a purified polynucleotide. The term “purified” polynucleotide refers to a polynucleotide that is substantially free from other nucleic acid sequences, such as and not limited to other chromosomal and extra-chromosomal DNA and RNA. Purified polynucleotides may be purified from a host cell. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain purified polynucleotides. The term also includes recombinant polynucleotides and chemically synthesized polynucleotides.
Yet another aspect of the invention is a plasmid or vector comprising a polynucleotide according to the invention. Preferably, the plasmid or vector is an expression vector. In a particular embodiment, the vector is a transfer vector for use in human gene therapy.
Still another aspect of the invention is a host cell comprising a polynucleotide of the invention or a plasmid or vector of the invention.
The host cells of the invention may be employed in a method of producing recombinant VWF and/or recombinant FVIII, which is part of this invention. The method comprises:
a) culturing host cells of the invention under conditions such that the VWF and/or FVIII is expressed; and
b) optionally recovering VWF and/or FVIII from the host cells or from the culture medium.
Degree and location of glycosylation or other post-translation modifications may vary depending on the chosen host cells and the nature of the host cellular environment. When referring to specific amino acid sequences, posttranslational modifications of such sequences are encompassed in this application.
“FVIII” as used in this application means a product consisting of the nonactivated form (FVIII). “FVIII” and “VWF” as used in this invention include proteins that have the amino acid sequence of native human FVIII and VWF respectively. It also includes proteins with a slightly modified amino acid sequence, for instance, a modified N-terminal end including N-terminal amino acid deletions or additions so long as those proteins substantially retain the activity of FVIII or VWF respectively. “FVIII” and “VWF” within the above definition also include natural allelic variations that may exist and occur from one individual to another. “FVIII” or “VWF” within the above definition further include variants of FVIII or VWF. Such variants differ in one or more amino acid residues from the wild type sequence. Examples of such differences may include truncation of the N- and/or C-terminus by one or more amino acid residues (e.g. 1 to 10 amino acid residues), or addition of one or more extra residues at the N- and/or C-terminus, e.g. addition of a methionine residue at the N-terminus, as well as conservative amino acid substitutions, i.e. substitutions performed within groups of amino acids with similar characteristics, e.g. (1) small amino acids, (2) acidic amino acids, (3) polar amino acids, (4) basic amino acids, (5) hydrophobic amino acids, (6) aromatic amino acids. Examples of such conservative substitutions are shown in the following table.
TABLE 1(1)AlanineGlycine(2)Aspartic acidGlutamic acid(3a)AsparagineGlutamine(3b)SerineThreonine(4)ArginineHistidineLysine(5)IsoleucineLeucineMethionineValine(6)PhenylalanineTyrosineTryptophane
The term “recombinant” means, for example, that the variant has been produced in a host organism by genetic engineering techniques. The FVIII or VWF variant of this invention is usually a recombinant variant.
Expression of the Proposed Variants:
The production of recombinant proteins at high levels in suitable host cells, requires the assembly of the above-mentioned modified cDNAs into efficient transcriptional units together with suitable regulatory elements in a recombinant expression vector, that can be propagated in various expression systems according to methods known to those skilled in the art. Efficient transcriptional regulatory elements could be derived from viruses having animal cells as their natural hosts or from the chromosomal DNA of animal cells. Preferably, promoter-enhancer combinations derived from the Simian Virus 40, adenovirus, BK polyoma virus, human cytomegalovirus, or the long terminal repeat of Rous sarcoma virus, or promoter-enhancer combinations including strongly constitutively transcribed genes in animal cells like beta-actin or GRP78 can be used. In order to achieve stable high levels of mRNA transcribed from the cDNAs, the transcriptional unit should contain in its 3′-proximal part a DNA region encoding a transcriptional termination-polyadenylation sequence. Preferably, this sequence is derived from the Simian Virus 40 early transcriptional region, the rabbit beta-globin gene, or the human tissue plasminogen activator gene.
The cDNAs are then integrated into the genome of a suitable host cell line for expression of FVIII or VWF. Preferably this cell line should be an animal cell-line of vertebrate origin in order to ensure correct folding, Gla-domain synthesis, disulfide bond formation, asparagine-linked glycosylation, O-linked glycosylation, and other post-translational modifications as well as secretion into the cultivation medium. Examples of other post-translational modifications are hydroxylation and proteolytic processing of the nascent polypeptide chain. Examples of cell lines that can be used are monkey COS-cells, mouse L-cells, mouse C127-cells, hamster BHK-21 cells, human embryonic kidney 293 cells, and hamster CHO-cells.
The recombinant expression vector encoding the corresponding cDNAs can be introduced into an animal cell line in several different ways. For instance, recombinant expression vectors can be created from vectors based on different animal viruses. Examples of these are vectors based on baculovirus, vaccinia virus, adenovirus, and preferably bovine papilloma virus.
The transcription units encoding the corresponding DNAs can also be introduced into animal cells together with another recombinant gene, which may function as a dominant selectable marker in these cells in order to facilitate the isolation of specific cell clones, which have integrated the recombinant DNA into their genome. Examples of this type of dominant selectable marker genes are Tn5 amino glycoside phosphotransferase, conferring resistance to geneticin (G418), hygromycin phosphotransferase, conferring resistance to hygromycin, and puromycin acetyl transferase, conferring resistance to puromycin. The recombinant expression vector encoding such a selectable marker can reside either on the same vector as the one encoding the cDNA of the desired protein, or it can be encoded on a separate vector which is simultaneously introduced and integrated into the genome of the host cell, frequently resulting in a tight physical linkage between the different transcription units.
Other types of selectable marker genes, which can be used together with the cDNA of the desired protein, are based on various transcription units encoding dihydrofolate reductase (dhfr). After introduction of this type of gene into cells lacking endogenous dhfr-activity, preferentially CHO-cells (DUKX-B11, DG-44) it will enable these to grow in media lacking nucleosides. An example of such a medium is Ham's F12 without hypoxanthine, thymidin, and glycine. These dhfr-genes can be introduced together with the coagulation factor cDNA transcriptional units into CHO-cells of the above type, either linked on the same vector or on different vectors, thus creating dhfr-positive cell lines producing recombinant protein.
If the above cell lines are grown in the presence of the cytotoxic dhfr-inhibitor methotrexate, new cell lines resistant to methotrexate will emerge. These cell lines may produce recombinant protein at an increased rate due to the amplified number of linked dhfr and the desired protein's transcriptional units. When propagating these cell lines in increasing concentrations of methotrexate (1-10000 nM), new cell lines can be obtained which produce the desired protein at very high rate.
The above cell lines producing the desired protein can be grown on a large scale, either in suspension culture or on various solid supports. Examples of these supports are micro carriers based on dextran or collagen matrices, or solid supports in the form of hollow fibres or various ceramic materials. When grown in cell suspension culture or on micro carriers the culture of the above cell lines can be performed either as a bath culture or as a perfusion culture with continuous production of conditioned medium over extended periods of time. Thus, according to the present invention, the above cell lines are well suited for the development of an industrial process for the production of the desired recombinant proteins.
The recombinant protein, which accumulates in the medium of secreting cells of the above types, can be concentrated and purified by a variety of biochemical and chromatographic methods, including methods utilizing differences in size, charge, hydrophobicity, solubility, specific affinity, etc. between the desired protein and other substances in the cell cultivation medium.
An example of such purification is the adsorption of the recombinant protein to a monoclonal antibody, which is immobilised on a solid support. After desorption, the protein can be further purified by a variety of chromatographic techniques based on the above properties.
It is preferred to purify the biologically active FVIII or VWF of the present invention to ≧80% purity, more preferably ≧95% purity, and particularly preferred is a pharmaceutically pure state that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, an isolated or purified biologically active FVIII or VWF of the invention is substantially free of other polypeptides except when a combination of FVIII and VWF should be administered.
The recombinant proteins described in this invention can be formulated into pharmaceutical preparations for therapeutic use. The purified proteins may be dissolved in conventional physiologically compatible aqueous buffer solutions to which there may be added, optionally, pharmaceutical excipients to provide pharmaceutical preparations.
Such pharmaceutical carriers and excipients as well as suitable pharmaceutical formulations are well known in the art (see for example “Pharmaceutical Formulation Development of Peptides and Proteins”, Frokjaer et al., Taylor & Francis (2000) or “Handbook of Pharmaceutical Excipients”, 3rd edition, Kibbe et al., Pharmaceutical Press (2000)). In particular, the pharmaceutical composition comprising the polypeptide variant of the invention may be formulated in lyophilized or stable soluble form. The polypeptide variant may be lyophilized by a variety of procedures known in the art. Lyophilized formulations are reconstituted prior to use by the addition of one or more pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution.
Formulations of the composition are delivered to the individual by any pharmaceutically suitable means of non-intravenous administration. Various delivery systems are known and can be used to administer the composition by any convenient route. Preferentially the compositions of the invention are formulated for subcutaneous, intramuscular, intraperitoneal, intracerebral, intrapulmonar, intranasal or transdermal administration, most preferably for subcutaneous, intramuscular or transdermal administration according to conventional methods. The formulations can be administered continuously by infusion or by bolus injection. Some formulations encompass slow release systems.
The modified biologically active FVIII and VWF polypeptides of the present invention are administered to patients in a therapeutically effective dose, meaning a dose that is sufficient to produce the desired effects, preventing or lessening the severity or spread of the condition or indication being treated without reaching a dose which produces intolerable adverse side effects. The exact dose depends on many factors as e.g. the indication, formulation, mode of administration and has to be determined in preclinical and clinical trials for each respective indication.
The pharmaceutical composition of the invention may be administered alone or in conjunction with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical.
Another aspect of the invention is the use of a VWF or a VWF combined with FVIII as described herein, of a polynucleotide of the invention, of a plasmid or vector of the invention, or of a host cell of the invention for the manufacture of a medicament for the treatment or prevention of a blood coagulation disorder. Blood coagulation disorders include but are not limited to hemophilia A and VWD, or FVII/FVIIa deficiency. Preferably these diseases are caused or congenital forms are aggravated by autoimmune antibodies against the respective coagulation factors. In a specific embodiment, the patients to be treated have inhibitor antibodies against factor VIII. Preferably, the treatment comprises human gene therapy.
The mode of administration is preferentially subcutaneous, but encompasses all extravascular routes of administration. This means that superficial administrations, i.e. non vascular as opposed to intravascular injections, would be most preferable to the patient. Most superficial administrations would be administration via epithelial surfaces (on the skin). Of special clinical utility would be an application via a patch. This topical administration requires uptake through the skin, which can be however quite marked, not only with superficial abrasions but also intact skin, and it may include eye drops and nasal applications. Administration via epithelial surfaces includes inhalation, which is suitable due to the extraordinary large surface covered with the protein, leading to rapid uptake and bypassing of the liver. Administration on epithelial surfaces includes dosage forms which are held in the mouth or under the tongue, i.e. are buccal or sublingual dosage forms, possibly even as chewing gum. Since the pH in the mouth is relatively neutral (as opposed to the acidic stomach milieu) this would be positive for a labile protein such as FVIII. Vaginal and even rectal administration might also be considered as some of the veins draining the rectum lead directly to the general circulation. Typically this is most helpful for patients who cannot take substances via the oral route, such as young children.
Intradermal injection (in the skin) would be a more invasive mode of administration, but still suitable for a treatment without assistance or even execution by trained personnel. Intradermal administration would be followed by subcutaneous injection (just under the skin). Typically uptake is quite substantial and can be increased by warming or massaging the injection area. Alternatively vasoconstriction can be achieved, resulting in the opposite behaviour, i.e. reducing the adsorption but prolonging the effect.
Even more invasive extravascular administration includes intramuscular delivery (into the body of the muscle). This might provide benefits by circumventing adipose tissue, but it is typically more painful that subcutaneous injections and especially with patients characterized by a deficient coagulation system, to be improved by the injection, there is the risk of tissue lesions, resulting in bleedings.
Independent of the degree of invasiveness, the VWF:FVIII complex might be loaded to biologically degradable particles, which can be designed to having a high affinity to epithelial surfaces and loaded with the VWF:FVIII complex in order to improve adsorption.
The invention also concerns a method of treating an individual suffering from a blood coagulation disorder such as hemophilia A or FVII/FVIIa deficiency, preferably these diseases are caused by or congenital forms are aggravated by autoimmune antibodies against the respective coagulation factors. The method comprises administering to said individual an efficient amount pharmaceutical composition comprising VWF or VWF in combination with FVIII as described herein. In another embodiment, the method comprises administering to the individual an efficient amount of the polynucleotide of the invention or of a plasmid or vector of the invention. Alternatively, the method may comprise administering to the individual an efficient amount of the host cells of the invention described herein.
One of the major problems in the therapy of hemophilia A is the development of neutralizing antibodies against FVIII. About 25% of these patients develop inhibitory antibodies, neutralizing the activity of FVIII. Accordingly FVIII substitution does not help any more to correct the patients' hemostasis. While such inhibitory antibodies typically are generated by the first couple of treatments, it is currently very difficult to predict which patients will suffer from this complication. Whether the particular FVIII concentrate used plays a role in this is a topic of controversial discussion in the scientific literature. From other proteins, designed to achieve a maximal immune response it is at least clear, that the application mode plays a crucial role, i.e. i.v. injections are typically less immunogenic than s.c. injections. However it was surprisingly found that if FVIII is formulated with VWF even if administered s.c. this formulation generates in hemophilia A mice less inhibitory antibodies than FVIII administered i.v. and certainly less inhibitory antibodies than an s.c. administration of FVIII.
Therefore another embodiment of the invention is the use of VWF for the manufacture of a medicament to treat VWD and/or hemophilia A wherein after extravascular co-administration with a pharmaceutical preparation of FVIII, less inhibitory antibodies against FVIII are generated as compared to when said pharmaceutical composition of FVIII is administered in the same concentration, dose and in the same mode of extravascular administration but without VWF.
For i.v. administration of FVIII there is an ongoing discussion in the scientific literature whether a formulation of FVIII with VWF might decrease the risk for the generation of inhibitory antibodies against FVIII. Our data (Example 10) point to a reduction in the generation of inhibitory antibodies against FVIII when a VWF formulated FVIII is administered.
In a preferred embodiment of the invention at least 15% less inhibitory antibodies are generated when a VWF preparation is extravascularly co-administered with a pharmaceutical preparation of FVIII, as compared to when said pharmaceutical composition of FVIII is administered in the same concentration, dose and in the same mode of extravascular administration but without VWF and wherein the titer of inhibitory antibodies is determined with the Bethesda assay (Goudemand J., Haemophilia, Vol. 13 Suppl. 5: 47-51, 2007; Ettingshausen C. E., Kreuz W., Haemophilia, Vol. 12 Suppl. 6: 102-106, 2006).
Preferably at least 25% less inhibitory antibodies are generated when a VWF preparation is extravascularly co-administered with a pharmaceutical preparation of FVIII, as compared to when said pharmaceutical composition of FVIII is administered in the same concentration, dose and in the same mode of extravascular administration but without VWF and wherein the titer of inhibitory antibodies is determined with the Bethesda assay (Goudemand J., Haemophilia, Vol. 13 Suppl. 5: 47-51, 2007; Ettingshausen C. E., Kreuz W., Haemophilia, Vol. 12 Suppl. 6: 102-106, 2006).
More preferably at least 50% less inhibitory antibodies are generated when a VWF preparation is extravascularly co-administered with a pharmaceutical preparation of FVIII, as compared to when said pharmaceutical composition of FVIII is administered in the same concentration, dose and in the same mode of extravascular administration but without VWF and wherein the titer of inhibitory antibodies is determined with the Bethesda assay.
Most preferably at least 75% less inhibitory antibodies are generated when a VWF preparation is extravascularly co-administered with a pharmaceutical preparation of FVIII, as compared to when said pharmaceutical composition of FVIII is administered in the same concentration, dose and in the same mode of extravascular administration but without VWF and wherein the titer of inhibitory antibodies is determined with the Bethesda assay.
In another preferred embodiment of the invention such VWF formulated FVIII is administered extravascularly to previously untreated patients, as the generation of inhibitory antibodies against FVIII most likely occurs during the initial doses of FVIII which a so far untreated hemophilia A patient receives.
In yet another preferred embodiment of the invention when administering FVIII together with VWF extravascularly, the administered VWF has a VWF:RCoF/VWF:Ag ratio which is less than 1:0.35, preferentially equal or less than 1:1.05.
Another preferred embodiment of the invention is the use of VWF for the manufacture of a medicament to treat VWD and/or hemophilia A, wherein the administered VWF is administered extravascularly and has a VWF:RCoF/VWF:Ag ratio which is less than 1:0.35 or preferentially less than 1:1.05.